Bruton's Tyrosine Kinase (BTK) Is Indispensable for Myeloma Cell Migration towards SDF-1 and Induction of Osteoclastogenesis and Osteolytic Bone Disease

Abstract 447

Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase of the TEC family of protein tyrosine kinases, is preferentially expressed in the hematopoietic cells and involved in B-lymphocytes survival and differentiation, and their migration towards SDF-1 gradient. BTK is also expressed in osteoclast precursors and plays indispensable role in osteoclastogenesis (Shinohara et al., Cell 2008). LFM-A13 is small molecule inhibitor of BTK and TEC, and can be tolerated even when animals are given daily dose of 100 mg/kg. The aims of the study were to investigate the effect of LFM-A13 on myeloma cell migration and growth in vitro, and on myeloma bone disease and tumor growth in the SCID-rab model. Our clinical gene expression profiling data indicate that in contrast to most myeloma cell lines, primary myeloma cells express BTK and that relative to normal plasma cells, BTK expression is upregulated in myeloma cells molecularly classified in the CD-1, CD-2, HY, LB and MF subtypes. Expression of BTK at the RNA and proteins levels in myeloma cells and osteoclast precursors was validated using qRT-PCR and Western Blot. To test effect on migration, myeloma cells expressing BTK and cell surface CXCR4 (n=5) were placed in the top of transwell inserts in the absence and presence of LFM-A13 (50 μM) and their migration towards SDF-1 (30 nM) was evaluated after 18 hours. SDF-1 induced migration of myeloma cells by >2.5 folds, an effect that was markedly inhibited by LFM-A13 (p<0.01). At similar concentration, this agent modestly reduced growth of BTK-expressing myeloma cells by 20%, assessed by MTT assay. LFM-A13 dose dependently inhibited osteoclast formation by 25% at 10 μM (p<0.0005) and by 80% at 40 μM (p<0.0001). In vivo, SCID-rab mice engrafted with a novel myeloma cell line, DAS, established through sequential passaging in this animal model (Xin et al., BJH 2007). DAS cells do not grow in culture, are molecularly classified as MF subtype and express high level of BTK. Upon establishment of myeloma growth, 2 weeks after tumor cell injection, hosts were intraperitoneally treated with 40 mg/kg LFM-A13 or vehicle (10 mice/group) twice a day for 3 weeks. Myeloma bone disease was evaluated by x-rays and analysis of bone mineral density (BMD). Tumor growth was analyzed by measurement of circulating human Ig and histologically. Whereas in the control group BMD of the implanted bone was reduced by 15±4% from pretreatment levels, it remained unchanged (0.8±4% change from pretreatment level) in the LFM-A13 treated hosts (p<0.01 versus control). X-ray radiographs revealed induction of osteolytic lesions in implanted bones of control hosts and that LFM-A13 effectively prevented these effects. LFM-A13 reduced the number of TRAP-expressing osteoclasts in myelomatous bones by >43% (p<0.004). At the end of the experiment, tumor burden was insignificantly lower in hosts treated with the LFM-A13 (Ig levels 42±15 and 19±5 μg/ml in control and LFM-A13 groups, respectively). We conclude that BTK is indispensable for SDF-1-indcued myeloma cell migration and stimulation of osteoclastogenesis, and that pharmacologic inhibition of BTK effectively inhibits myeloma-induced bone resorption and may interrupt with myeloma cell homing and metastasis to bone.